Immunoglobulin G1 enzyme-linked immunosorbent assay for diagnosis of Johne's Disease in red deer (Cervus elaphus)

Abstract

This study was designed to develop a customized enzyme-linked immunosorbent assay (ELISA) for the serodiagnosis of Johne's disease (JD) in farmed deer. Two antigens were selected on the basis of their superior diagnostic readouts: denatured purified protein derivative (PPDj) and undenatured protoplasmic antigen (PpAg). ELISA development was based on the antigen reactivity of the immunoglobulin G1 (IgG1) isotype, which is a highly specific marker for mycobacterial disease seroreactivity in deer. Sensitivity estimates and test parameters were established using 102 Mycobacterium paratuberculosis-infected animals from more than 10 deer herds, and specificity estimates were determined using 508 uninfected animals from 5 known disease-free herds. A receiver-operated characteristic analysis determined that at a cut point of 50 ELISA units, there was a specificity of 99.5% and sensitivities of 84.0% with PPDj antigen, 88.0% with PpAg, and 91.0% when the antigens were used serially in a composite test. Estimated sensitivity was further improved using recombinant protein antigens unique for M. paratuberculosis, which identified infected animals that were unreactive to PPDj or PpAg. While 80% of animals that were seropositive in the IgG1 ELISA had detectable histopathology, the assay could also detect animals with subclinical disease. The test was significantly less sensitive (75%) for animals that were culture positive for M. paratuberculosis but with no detectable pathology than for those with pathological evidence of JD (>90%). When the IgG1 ELISA was used annually over a 4-year period in a deer herd with high levels of clinical JD, it eliminated clinical disease, increased production levels, and reduced JD-related mortality.

FIG. 1.

ROC curve analysis of IgG1 ELISA. Estimated sensitivity and specificity values using complex M. paratuberculosis protein antigens. Serum samples from 102 deer with confirmed M. paratuberculosis infection, as well as 508 samples obtained from deer herds with no prior history of JD, were assayed by IgG1 ELISA using PPDj (A) or PpAg (B) as the target antigen. Data were entered into a ROC analysis program; numerals refer to the calculated ELISA cut points and estimated test sensitivity values corresponding to 100% estimated test specificity for each antigen.

FIG. 2.

IgG1 antibody responses in M. paratuberculosis-infected and noninfected deer against a panel of recombinant M. paratuberculosis antigens. Sera from 10 M. paratuberculosis-infected deer (black bars) and 10 noninfected deer (white bars) were screened by IgG1 ELISA against a range of recombinant M. paratuberculosis antigens. Responses against complex protein antigens (PpAg and PPDj) are shown for comparison. Data represent mean EU ± standard errors of the mean.

FIG. 3.

Longitudinal changes in the proportion of test-positive and test-negative adult deer over a 4-year period following implementation of an IgG1-based ELISA test-and-cull management strategy. Regular serological screening for M. paratuberculosis infection was undertaken on a deer herd with a known history of JD. Screening commenced in 2002 (with an initial cut point of 100 EU) and proceeded over a 4-year period (with the cut point dropping to 50 EU after 6 months); test-positive animals were culled from the herd upon testing positive. Data represent longitudinal changes in the number of test-positive animals (gray bars) against test-negative animals (black bars) (data are shown for adult hinds only; data for stags not shown).

FIG. 4.

Longitudinal changes in the number of IgG1 ELISA-positive young deer following implementation of an ELISA-based test-and-cull management strategy. Young deer (fawns) were screened by IgG1 ELISA to detect M. paratuberculosis infection at two ages, 6 to 8 months (gray bars) and 12 to 15 months (black bars). Data represent the number of fawns testing positive at each time point, following the implementation of a test-and-cull management strategy in 2002.